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Abstract

Our previous studies indicated that Met5-enkephalin (ME)-induced protection of cardiomyocytes occurs via transactivation of EGFR with subsequent activation of the phosphoinositol-3-kinase (PI3K) / Akt pathway. In vivo, tolerance to myocardial ischemia is gender-dependent. We tested the hypothesis that there is a gender difference in ME-elicited signaling. PND7 murine cardiomyocytes were isolated by collagenase digestion, cultured for 24–72 hr, and subjected to 90 min hypoxia and 180 min reoxygenation at 37C (n=5–7 replicates). PI3K / Akt signaling was interrogated using pharmacologic inhibitors and small interfering RNA (siRNA). Cell death was assessed by propidium iodide. More than 300 cells were examined for each treatment. Data are presented as mean ± SEM. There were no gender differences in basal levels of total Akt as assessed by immunoblot. ME (100 μM), given 15 min before hypoxia, elicited comparable protection in both genders. Wortmannin and the non-selective Akt-inhibitor IV, given 15 min after ME, completely abolished ME-induced protection in male cardiomyocytes but merely attenuated protection in female cardiomyocytes. Isoform-selective knock-down of Akt showed that in males siRNA against Akt1/2 completely abolished ME-induced cardioprotection whereas siRNA against Akt3 only attenuated protection ~ 40%; in contrast, in females siRNAs against both Akt1/2 and Akt3 demonstrated near-complete elimination of ME-induced cardioprotection. We conclude that (1) there is no gender difference in the degree of ME-induced protection, and (2) there is a gender difference in cardioprotective signaling pathways after administration of ME: ME-induced cardioprotection in males utilizes a PI3K -Akt1/2 pathway, and in females utilizes PI3K and both Akt1/2 and Akt3. Further, incomplete loss of protection in females following blockade of PI3K suggests additional factors may facilitate the maintenance or function of activated Akt.